AIDS (acquired immunodeficiency syndrome) is one of the leading causes of death in the developing world, its spread reaching pandemic proportions. In 1984, the etiologic agent of AIDS was discovered as the Human Immunodeficiency Virus (HIV), being a retrovirus which is a member of the lentivirus subfamily. The Lentiviridae include non-oncogenic retroviruses which usually infect cells of the immune system, particularly macrophages and T cells, causing persistent infections in diseases with long incubation periods and cytopathic effects in infected cells, such as syncytia and cell death. Lentiviral infections are not cleared by the immune system, and lead to accumulated immunologic damage over a period of many years.
HIV which is a member of the Lentiviridae is a retrovirus, i.e. containing an RNA genome and reverse transcriptase activity, and therefore, during its growth cycle, HIV copies its RNA into proviral DNA, which is able to integrate into the chromosomal DNA of the host cell (provirus). Due to its retroviral nature and small size of its genome, HIV replication is strongly dependent on the host's cell machinery. Thus, HIV uses the transcriptional and translational machinery of the host to express viral RNA and proteins and to finally release mature viruses from the cell by budding from the cytoplasmic membrane. In the case of HIV, viral replication results in the death of host's helper T cell, which leads to a state of severe immunodeficiency (AIDS), to the development of various malignancies and opportunistic infections, and ultimately to the death of the infected organism.
Besides the “usual” genes of the HIV genome such as env (encoding the virus' envelope protein), gag (encoding the internal proteins responsible for forming the capsid- and nucleocapsid structures) and pol (encoding the enzymes reverse transcriptase, integrase and protease), the transcriptional transactivator (tat) and the regulator of viral expression (rev) genes produce small non-virion proteins which are essential for viral replication. Also several genes which are not implicated in viral expression are encoded by HIV such as vif, vpr, vpu and nef.
The treatment of HIV disease has been significantly advanced by recognizing that the HIV life cycle can be interfered with at many levels by for example inhibiting virus' reverse transcription, inhibiting its protease activity or fusion. Accordingly, three major classes of drugs have been developed: reverse transcriptase inhibitors, protease inhibitors and fusion inhibitors, and currently, there are about 25 antiretroviral drugs approved to treat individuals infected with HIV. Furthermore, it is known that the combination of different drugs with specific activities against different biochemical functions of the virus (combination therapy) can help reducing the rapid development of drug resistant viruses that was observed in response to single drug treatments.
However, even the current “highly active anti-retroviral therapy” (HAART), which is based on a treatment of infected persons with a combination of antiviral drugs from at least two of the above-referenced drug classes, and which to date is the only efficacious treatment to reduce progression and spread of AIDS, is associated with several drawbacks and limitations when used long-termed, such as adherence to a complex dosing regimen, side effect toxicity and elevated costs; see for example Richman, Nature 410 (2001), 995-1001. Additionally, the high genetic and antigenic variability of HIV, due to the high mutation rate of its genome, in combination with inadequate compliance, is responsible for resistance to HAART drugs, because of which although the use of HAART has greatly reduced the number of deaths due to HIV/AIDS, complete viral suppression was not achieved.
In addition to the development of inhibitors of the “essential” viral enzymes (reverse transcriptase, protease and integrase), research is currently focussed on targeting HIV accessory proteins. For the HIV accessory proteins Vif, Vpu, Vpr, and Nef the precise biochemical mechanisms are still under investigation, however, there is increasing evidence to suggest that none of these proteins has catalytic activity on its own, but rather, they appear to function as “adapter molecules” that connect other viral or cellular factors to various cellular pathways.
One of the four accessory proteins, the virion infectivity factor (Vif), is a small basic phosphoprotein with a molecular mass of 23 kDa and composed of 192 amino acids, which is synthesized in a Rev-dependent manner during the late stages of virion production. Homologs of Vif exist in all lentiviruses, with the only exception being equine infectious anaemia virus (EIAV), see Oberste & Gonda, Virus Genes 6 (1992), 95-102, and there is significant conservation among vif open reading frames of the different lentiviruses; see Sonigo et al., Cell 42 (1985), 369-382.
In case of HIV, Vif is generally required for viral replication in primary T cells. Furthermore, it is known to counteract suppression of HIV replication mediated by a host protein, i.e. the human apolipoprotein B mRNA-edlting enzyme-catalytic-polypeptide-like-3G (APOBEC3G), which is a member of the APOBEC cytodine deaminase family of enzymes. APOBEC3G is known to be packaged into retroviral virions (infectious mature virus particles) and to deaminate deoxycytidine to deoxyuridine in newly synthesized viral minus-strand DNA, thereby inducing G-to-A hypermutation. The interaction of Vif with APOBEC3G in the virus producing cell prevents APOBEC3G from being incorporated into virions and thus prevents APOBEC3G from acting on newly synthesized HIV cDNA. To the contrary, Vif was shown to interact with cellular proteins Cul5, ElonginB, ElonginC, and Rbx1 to form an E3 ubiquitin ligase complex for APOBEC3G ubiquitination (see, for example, Yu at al., Science 302 (2003), 1056-1060), thereby upon binding to APOBEC3G inducing its ubiquitination and proteosomal degradation and finally the elimination of APOBEC3G from cells, which enables HIV to produce infectious viruses.
Vif was also shown to interact with the Src tyrosine kinases Fyn and Hck resulting in a reduction of their catalytic activities; see for example Hassaine et al., J. Biol. Chem. 276 (2001), 16885-16893; and to interact with the zinc finger protein inhibiting NF-KB; see Feng et al., J. Virol. 78 (2004), 10574. Since the protein interaction network of Vif in the host cell being responsible for the functional role of the viral accessory protein is only partially understood, recently novel functions in cell cycle regulation of infected cells were described by Wang et al., Virology 359 (2007), 243-252.
However, none of the human proteins identified so far to interact with HIV accessory proteins could be shown to be suitable as a target for anti-HIV drug development. Thus, there is still a need for providing anti-HIV drug targets that supplement the “essential” proteins, reverse transcriptase, protease and integrase, in the combined anti-HIV therapy or provide an alternative thereto.